A Simple Method for Quantification of Protochlorophyllide in Etiolated Arabidopsis Seedlings.

Etiolated seedlings accumulate the chlorophyll biosynthesis intermediate protochlorophyllide (Pchlide) and measuring Pchlide can be important for characterizing photomorphogenic mutants that may be affected in chloroplast development. In this chapter we outline a simple and sensitive method for quantifying Pchlide in extracts of Arabidopsis seedlings using fluorescence spectroscopy. This method can be easily adapted to study chloroplast development in a wide range of plant species.

Light piping activates chlorophyll biosynthesis in the under-soil hypocotyl section of bean seedlings.

Protochlorophyllide (Pchlide), protochlorophyll (Pchl) and chlorophyll (Chl) contents, their distribution and native arrangements were studied in under-soil hypocotyl segments of 4-, 7- and 14-day-old bean (Phaseolus vulgaris L. cv. Magnum) seedlings. The plants were grown in general potting soil under natural illumination conditions in pots. For sample collection, the pots were transferred into dark-room where all manipulations were done under dim green light. The pigments were extracted with acetone; phase separation was used to identify the Pchl contents. Fluorescence microscopic studies were done and 77K fluorescence emission spectra were recorded. Using a special setup of a spectrofluorometer, the vertical light piping properties of the above-soil shoots were measured. The segments in the 5-7 cm deep soil region contained Pchlide and Pchl in 4- and 7-day-old seedlings and the segments towards the soil surface contained Chl in increasing amounts. In parallel with the pith degradation of hypocotyls, the Chl content of elder seedlings increased in the deeper under-soil segments. These results prove that the tissue structure of the shoot ensures light piping thus greening process and chloroplast formation can take place even in under-soil organs not directly exposed to light.

Methods for analysis of photosynthetic pigments and steady-state levels of intermediates of tetrapyrrole biosynthesis.

Tetrapyrroles and carotenoids are required for many indispensable functions in photosynthesis. Tetrapyrroles are essential metabolites for photosynthesis, redox reaction, and detoxification of reactive oxygen species and xenobiotics, while carotenoids function as accessory pigments, in photoprotection and in attraction to animals. Their branched metabolic pathways of synthesis and degradation are tightly controlled to provide adequate amounts of each metabolite (carotenoids/tetrapyrroles) and to prevent accumulation of photoreactive intermediates (tetrapyrroles). Many Arabidopsis mutants and transgenic plants have been reported to show variations in steady-state levels of tetrapyrrole intermediates and contents of different carotenoid species. It is a challenging task to determine the minute amounts of these metabolites to assess the metabolic flow and the activities of both pigment-synthesising and degrading pathways, to unravel limiting enzymatic steps of these biosynthetic pathways, and to characterise mutants with accumulating intermediates. In this chapter, we present a series of methods to qualify and quantify anabolic and catabolic intermediates of Arabidopsis tetrapyrrole metabolism, and describe a common method for quantification of different plant carotenoid species. Additionally, we introduce two methods for quantification of non-covalently bound haem. The approach of analysing steady-state levels of tetrapyrrole intermediates in plants, when applied in combination with analyses of transcripts, proteins, and enzyme activities, enables the biochemical and genetic elucidation of the tetrapyrrole pathway in wild-type plants, varieties, and mutants. Steady-state levels of tetrapyrrole intermediates are only up to 1/1,000 of the amounts of the accumulating end-products, chlorophyll, and haem. Although present in very low amounts, the accumulation and availability of tetrapyrrole intermediates have major consequences on the physiology and activity of chloroplasts due to their additional photoreactive and possible signalling functions. Although adjusted for Arabidopsis tetrapyrrole metabolites, the presented methods can also be applied for analysis of cyanobacterial and other plant tetrapyrroles.

Biological variability in the ratios of protochlorophyllide forms in leaves and epicotyls of dark-grown pea (Pisum sativum L.) seedlings (a statistical method to resolve complex spectra).

Low-temperature (77K) fluorescence emission spectra of 100 dark-grown pea (Pisum sativum L.) seedlings of various ages were measured. The spectra of the 100 leaf samples were collected into a separate data group and those of epicotyls formed another one. This group was divided into three sub-groups as spectra of uppermost, middle and lowermost 3 cm sections. Further sub-groups were formed on the basis of the ages of the plants. The spectra were normalized to their total integral values (within the 580-780 nm region) then the AVERAGE (arithmetic mean function) and AVEDEV (average of the absolute deviations of data points of their mean function) spectra were calculated. Very sharp bands were found in the AVEDEV spectra. Even the strongly overlapped 629 and 636 nm emission bands appeared as separate peaks, due to the decrease of their half-bandwidth values in the AVEDEV function. Both types of spectra were resolved into Gaussian components. The results showed that the variabilities of the 633 and 655 nm protochlorophyllide forms were similar in the leaves. In epicotyls, the protochlorophyllide forms had different variabilities, especially in the middle sections. The most variable was the amplitude of the 636 nm band and the variabilities of the 629 and 655 nm bands were smaller but still remarkable. The calculation of AVEDEV spectra is an effective method to study the biological variability and spectral resolution of biological samples containing chromophores with multiple spectral properties.

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana.

Shortly after the release of singlet oxygen ((1)O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of (1)O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of (1)O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of (1)O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all (1)O2-responsive genes. Retrograde control of (1)O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.

Occurrence of chlorophyll precursors in leaves of cabbage heads--the case of natural etiolation.

In the interior of cabbage (Brassica oleracea) heads (kale, white cabbage, Brussels sprouts), natural leaf etiolation takes place due to a limited light access and chlorophyll biosynthesis is inhibited in a consequence. Instead, apart from carotenoids, whose biosynthesis is not light-dependent, chlorophyll precursors accumulate, mainly protochlorophyllide and to a smaller extent also chlorophyllides a and b. Protochlorophyllide was also detected in green, light-exposed leaves of heads of all the investigated cabbage varieties. Protochlorophyll was not found in the investigated leaves. The analysis of xanthophylls composition showed that the central leaves of kale and white cabbage heads contain relatively high amounts of trans-neoxanthin and lutein epoxide which are found only in trace amounts in green leaves. This is the first systematic study on natural occurrence of chlorophyll biosynthesis precursors in different cabbage varieties.

Identification of spectral forms of protochlorophyllide in the region 670-730 nm.

Dark-grown leaves of three different species, maize, wheat, pea and a pea mutant (lip1) have been used to study protochlorophyllide (Pchlide) spectral forms. As a comparison also pea epicotyls were used. Different native forms of Pchlide were identified using the variation in the spectral properties of the plant material and the second derivatives of the 77 K fluorescence excitation and emission spectra. The spectral forms were further characterised by Gaussian deconvolution. In addition to short-wavelength and long-wavelength forms the area between 660 and 730 nm was shown to contain, together with some vibrational bands, five far-red Pchlide forms. They had pairs of excitation and emission peaks at 658 and 666 nm, 668 and 680 nm, 677 and 690 nm, 686 and 698 as well as 696 and 728 nm, respectively. The presence of several far-red Pchlide forms in dark-grown leaves gave evidence for additional aggregated states of Pchlide under native conditions.

Fluorescence lifetimes of protochlorophyllide in plants with different proportions of short-wavelength and long-wavelength protochlorophyllide spectral forms.

Dark-grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild-type pea (Pisum sativum) and its light-independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low-temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild-type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide(650-656) form but also for the short-wavelength Pchlide forms. A fast component in the range 0.3-0.8 ns and a slow component in the range 5.1-7.1 ns were present in all samples, but the values varied, depending on species. The long-wavelength Pchlide(650-656) form had a slow component with a lifetime between 5.1 and 6.7 ns, probably reflecting the fluorescence from aggregated Pchlide. The short-wavelength Pchlide(628-633) form had values of the slow component varying between 6.2 and 7.1 ns. This represents a monomeric but probably protein-bound Pchlide form because the free Pchlide in solution has a much longer lifetime around 10 ns at 77 K. The contribution of different Pchlide forms to the measured lifetime values is discussed.

Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state.

Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH:protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli. The results obtained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R)-protochlorophyllide] demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site. The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme. At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide. We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.

Protochlorophyllide b does not occur in barley etioplasts.

Barley (Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection < 1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH :protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7-hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80-84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.

Parasitic apicomplexans harbor a chlorophyll a-D1 complex, the potential target for therapeutic triazines.

Ultrastructural evidence is presented for the presence of plastid-like organelles in Toxoplasma gondii, Sarcocystis muris, Babesia ovis, and Plasmodium falciparum. In addition, it was shown that merozoites of T. gondii contain protochlorophyllidae a and traces of chlorophyll a bound to the photosynthetic reaction centers I PS I and PS II. A psbA gene was isolated from merozoites of S. muris by the polymerase chain reaction (PCR). Partial sequencing of the PCR product revealed that the herbicide-binding region is highly conserved. Therefore, it is likely that the sensitivity of apicomplexans to the herbicide toltrazuril depends on the interaction of the herbicide with the D1 protein of the photosynthetic reaction center of the parasite's organelles.

Spectrofluorometric estimation of intermediates of chlorophyll biosynthesis: protoporphyrin IX, Mg-protoporphyrin, and protochlorophyllide.

A highly sensitive spectrofluorometric method for quantitative estimation of certain precursors of chlorophyll biosynthesis from the mixtures of plant tetrapyrroles having overlapping fluorescence emission spectra is developed. At room temperature (293 degrees K) protoporphyrin IX is monitored from its emission maximum, 633 nm, when excited at 400 nm (E400/F633). Protochlorophyllide is estimated at 638 nm, while being excited at 440 nm (E440/F638). Mg-protoporphyrin+Mg-protoporphyrin monoester pool has emission around 589-592 nm. Therefore the integration value of the emission band that extends from 580 to 610 nm is taken to calibrate its concentration. This spectrofluorometric method designed for the determination of protoporphyrin IX, esterified and nonesterified Mg-protoporphyrin pool, and protochlorophyllide is far superior to available spectrophotometric methods and estimates as low as 1 nM concentration of plant pigments. As minute quantities of individual pigments can be quantitatively analyzed from their mixtures, this method eliminates analytical uncertainties due to recovery losses caused by chromatography. However, only dilute samples can be estimated by this spectrofluorometric method as the quantitative relation between fluorescence and concentration deviates from linearity at high, i.e., above 150 nM, concentrations of pigment to be quantified.

Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley.

A photoactive protochlorophyllide-protein complex with absorbance and fluorescence maxima at 648 and 653 nm was detected in greening barley leaves without any re-darkening. The variations of the amplitudes of the absorbance and the fluorescence of the photoactive protochlorophyllide with greening time at two different light intensities indicate a close relationship between the rate of chlorophyll synthesis and the amount of the complex during the first hours. The chlorophyllide resulting from photoreduction during greening has an absorbance maximum at 684 nm, which shifts towards a shorter wavelength within a few seconds, indicating rapid liberation of the pigment from the enzyme. We conclude that chlorophyll accumulation proceeds through continuous regeneration and phototransformation of the photoactive complex.

Chloroplast biogenesis. Detection of monovinyl protochlorophyll(ide) b in plants.

The occurrence of protochlorophyllide b and protochlorophyllide b phytyl ester in green plants is described. The chemical structure of protochlorophyllide b phytyl ester was established by proton nuclear magnetic resonance, fast atom bombardment mass spectroscopic analysis, and chemical derivatization coupled to electronic spectroscopic analysis. The macrocycles of protochlorophyll(ide) b are identical to those of conventional protochlorophyll(ide) except for the presence of a formyl group instead of a methyl group at position 3 of the macrocycles. They differ from chlorophyll(ide) b by the presence of an oxidized double bond at positions 7 and 8 of the macrocycles. The trivial name protochlorophyll(ide) b is proposed to differentiate these two tetrapyrroles from conventional protochlorophyll(ide), which in turn will be referred to as protochlorophyll(ide) a. Protochlorophyll(ide) b appears to be widely distributed in green plants. Its molar extinction coefficients in 80% acetone and diethyl ether are reported. The impact of this discovery on the heterogeneity of the chlorophyll a and b biosynthetic pathways is discussed.

Fluorescence properties of the envelope membranes from spinach chloroplasts. Detection of protochlorophyllide.

At 77 K, under excitation at 440 nm, two major fluorescence emission peaks were observed in envelope membranes from spinach chloroplasts at 636 and 680 nm. A narrow range of wavelengths around 440 nm and a wider range of wavelengths between 390 and 440 nm, respectively, were responsible for excitation of the 636 and 680 nm fluorescence emissions which, in marked contrast with thylakoid fluorescence emission, were devoid of any exciting components between 460 and 500 nm. In acetonic extract of envelope membranes, two fluorescence emission peaks were observed at 635 and 675 nm. After extraction of the acetonic solution by nonpolar solvents (petroleum ether or hexane), the 675 nm fluorescence emission was partitioned between the polar and nonpolar phases whereas the 635 nm fluorescence emission was solely recovered in the polar phase. All together, the results obtained suggest that envelope membranes contain low amounts of pigments having the absorption and fluorescence spectroscopic properties, together with the behavior in polar/nonpolar solvents, of protochlorophyllide and chlorophyllide. In addition, modulation of the level of fluorescence at 636 and 680 nm could be obtained by addition of NADPH to envelope membranes under illumination. The presence of protochlorophyllide in chloroplast envelope membranes together with its possible photoconversion into chlorophyllide could have major implication for the understanding of chlorophyll biosynthesis in mature chloroplasts.

Chloroplast biogenesis: quantitative determination of monovinyl and divinyl Mg-protoporphyrins and protochlorophyll(ides) by spectrofluorometry.

General equations which permit the determination of the amounts of any two closely related fluorescent compounds which can be distinguished by 77 degrees K but not by 293 degrees K spectrofluorometry have been described. This was achieved in the presence or absence of a third interfering compound, without prior separation of the fluorescent species. The adaptation of the generalized equations to the determination of the amounts of monovinyl (MV) and divinyl (DV) Mg-protoporphyrins or of MV and DV protochlorophyll(ides) in the presence or absence of Mg-Protos [Mg-protoporphyrin IX (Mg-Proto), Mg-Proto monoester, Mg-Proto diester or a mixture of those three tetrapyrroles] interference, was then demonstrated over a wide range of MV/DV tetrapyrrole proportions. These equations are likely to be very useful for the study of the intermediary metabolism of the monovinyl and divinyl chlorophyll biosynthetic routes in plants.

Mg-2,4-divinyl pheoporphyrin a5: the product of a reaction catalyzed in vitro by developing chloroplasts.

The major product of an aerobic reaction mixture containing developing chloroplasts, Mg-protoporphyrin IX, S-adenosylmethionine, and other cofactors was isolated and purified. Structural studies using nuclear magnetic resonance confirmed earlier reports, based on fluorescence and absorption spectra, that this compound is Mg-2,4-divinyl pheoporphyrin a5. The molecular weight determined by secondary-ion mass spectroscopy further confirmed the assigned structure. Absorption and fluorescence spectra indicate that this compound is identical to that reported previously by various workers in less-purified biological extracts. The nuclear magnetic resonance spectrum of the Mg-free base also supports the assigned structure.

Chloroplast biogenesis. Detection of divinyl protochlorophyllide in higher plants.

It is shown that the protochlorophyllide pool of etiolated higher plants is made up of both monovinyl and divinyl protochlorophyllide. Although the two pigments exhibited similar emission maxima, they were distinguishable by their Soret excitation maxima, which were found at 436 to 437 and 443 to 444 nm, respectively, in ether at 77 K. The two pigments were partially separated on thin layers of polyethylene. They were shown to be accompanied by two unknown fluorescent compounds. The latter were designated compound (E451 F626) and compound (E453 F640) where E refers to the Soret excitation maxima and F to the fluorescence emission maxima of the two unknown compounds. Furthermore, it was shown under several different growth conditions that divinyl protochlorophyllide constituted the major component of the protochlorophyllide pool.